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Differentiation of Human Wharton Jelly Mesenchymal Stem Cells into Germ-Like Cells; emphasis on evaluation of Germ-long non-coding RNAs

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Abstract

Background

The proliferation and differentiation of stem cells into Germ-Like Cells (GLCs) is mediated by several growth factors and specific genes, of which some are related to long non-coding RNAs (lncRNAs). We have developed a modified differentiation process and identified a panel of GermlncRNAs related to GLCs.

Methods

Human Wharton Jelly Mesenchymal Stem Cells were treated with 25 ng/ml Bone Morphogenetic Protein (BMP)-4 and 10− 5 M all-trans retinoic acid to differentiate them into germ-like cells. To confirm the differentiation, changes in the expression of Oct-4, C-kit, Stella, and Vasa genes were assessed using quantitative Real-Time PCR (qPCR) and immunocytochemistry. QPCR was also used before and after differentiation to evaluate the changes in a lncRNA panel, using a 96-well array. Statistical analysis of the data was performed by SPSS 21.

Results

After 21 days of induction, the HWJ-MSCs derived germ-like cells were formed. Also, qPCR and immunocytochemistry showed that the pluripotent Oct4 marker was expressed in the undifferentiated HWJ-MSCs, but its expression gradually decreased in the differentiated cells. C-kit was expressed on days 7, 14, and 21 of differentiation. Both GLC markers of Stella and Vasa genes/proteins were present only in differentiated cells. Of the 44 lncRNA genes array, 36 of them showed an increase and eight genes showed a decrease.

Conclusion

Our study showed that BMP4 and RA are effective in inducing HWJ-MSCs differentiation into GLCs. In addition, our study for the first time showed changes in the lncRNAs expression during the differentiation of HWJ-MSCs into GLCs by using BMP4 and RA.

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References

  1. Adamson GD, de Mouzon J, Lancaster P, Nygren K-G, Sullivan E, Zegers-Hochschild F et al (2006) World collaborative report on in vitro fertilization, 2000. Fertil Steril 85(6):1586–1622

    Article  PubMed  Google Scholar 

  2. Hou J, Yang S, Yang H, Liu Y, Liu Y, Hai Y et al (2014) Generation of male differentiated germ cells from various types of stem cells. Reproduction 147(6):R179–R88

    Article  CAS  PubMed  Google Scholar 

  3. Anawalt BD (2013) Approach to male infertility and induction of spermatogenesis. J Clin Endocrinol Metab 98(9):3532–3542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Abrao MS, Muzii L, Marana R (2013) Anatomical causes of female infertility and their management. Int J Gynecol Obstet 123:S18–S24

    Article  Google Scholar 

  5. Sharpe RM (2010) Environmental/lifestyle effects on spermatogenesis. Philos Trans R Soc B 365(1546):1697–1712

    Article  CAS  Google Scholar 

  6. Anderson JE, Farr SL, Jamieson DJ, Warner L, Macaluso M (2009) Infertility services reported by men in the United States: national survey data. Fertil Steril 91(6):2466–2470

    Article  PubMed  Google Scholar 

  7. Morena AR, Boitani C, Pesce M, De Felici M, Stefanini M (1996) Isolation of highly purified type A spermatogonia from prepubertal rat testis. J Androl 17(6):708–717

    CAS  PubMed  Google Scholar 

  8. Leatherman J (2013) Stem cells supporting other stem cells. Front Genet 4:257

    Article  PubMed  PubMed Central  Google Scholar 

  9. Volarevic V, Bojic S, Nurkovic J, Volarevic A, Ljujic B, Arsenijevic N et al (2014) Stem cells as new agents for the treatment of infertility: current and future perspectives and challenges. BioMed Res Int 2014

  10. Chao KC, Chao KF, Fu YS, Liu SH (2008) Islet-like clusters derived from mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS ONE 3(1):e1451

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ayatollahi M, Talaei-Khozani T, Razmkhah M (2016) Growth suppression effect of human mesenchymal stem cells from bone marrow, adipose tissue, and Wharton’s Jelly of umbilical cord on PBMCs. Iran J Basic Med Sci 19(2):145–153

    PubMed  PubMed Central  Google Scholar 

  12. Zhang H, Zheng W, Shen Y, Adhikari D, Ueno H, Liu K (2012) Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries. Proc Natl Acad Sci 109(31):12580–12585

  13. Phillips BT, Gassei K, Orwig KE (2010) Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc B 365(1546):1663–1678

    Article  CAS  Google Scholar 

  14. Ganesan G, Rao SM (2008) A novel noncoding RNA processed by Drosha is restricted to nucleus in mouse. RNA 14(7):1399–1410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Trovero MF, Rodríguez-Casuriaga R, Romeo C, Santiñaque FF, François M, Folle GA et al (2020) Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis. RNA Biol 17(3):350–365

    Article  CAS  PubMed  Google Scholar 

  16. Liu L, Fang F (2022) Long noncoding RNA mediated regulation in human embryogenesis, pluripotency, and reproduction. Stem Cells Int 2022:8051717

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lee T-L, Xiao A, Rennert OM (2012) Identification of novel long noncoding RNA transcripts in male germ cells. Germline development. Springer, New York, pp 105–114

    Google Scholar 

  18. Lee TL, Pang ALY, Rennert OM, Chan WY (2009) Genomic landscape of developing male germ cells. Birth Defects Res Part C 87(1):43–63

    Article  CAS  PubMed  Google Scholar 

  19. Chen Q, Meng X, Liao Q, Chen M (2019) Versatile interactions and bioinformatics analysis of noncoding RNAs. Brief Bioinform 20(5):1781–1794

    Article  CAS  PubMed  Google Scholar 

  20. Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43(6):904–914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Latifpour M, Shakiba Y, Amidi F, Mazaheri Z, Sobhani A (2014) Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells. Avicenna J Med Biotechnol 6(4):218

    PubMed  PubMed Central  Google Scholar 

  22. Yan G, Fan Y, Li P, Zhang Y, Wang F (2015) Ectopic expression of DAZL gene in goat bone marrow-derived mesenchymal stem cells enhances the trans‐differentiation to putative germ cells compared to the exogenous treatment of retinoic acid or bone morphogenetic protein 4 signalling molecules. Cell Biol Int 39(1):74–83

    Article  CAS  PubMed  Google Scholar 

  23. Hua J, Pan S, Yang C, Dong W, Dou Z, Sidhu KS (2009) Derivation of male germ cell-like lineage from human fetal bone marrow stem cells. Reprod Biomed Online 19(1):99–105

    Article  CAS  PubMed  Google Scholar 

  24. Jafarpour Z, Soleimani M, Hosseinkhani S (2018) Efficient production of hepatocyte-like cells from human-induced pluripotent stem cells by optimizing growth factors. Int J Organ Transpl Med 9(2):77

    CAS  Google Scholar 

  25. Jaafarpour Z, Soleimani M, Hosseinkhani S, Karimi MH, Yaghmaei P, Mobarra N et al (2016) Differentiation of definitive endoderm from human induced pluripotent stem cells on hMSCs feeder in a defined medium. Avicenna J Med Biotechnol 8(1):2

    PubMed  PubMed Central  Google Scholar 

  26. Amidi F, Hoseini MA, Nia KN, Habibi M, Kajbafzadeh AM, Mazaheri Z et al (2015) Male germ-like cell differentiation potential of human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid. Iran J Basic Med Sci 18(4):325

    PubMed  PubMed Central  Google Scholar 

  27. Afsartala Z, Rezvanfar MA, Hodjat M, Tanha S, Assadollahi V, Bijangi K et al (2016) Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro. Vitro Cell Dev Biol 52(10):1060–1071

    Article  CAS  Google Scholar 

  28. Hussain SR, Naqvi H, Ahmed F, Babu SG, Bansal C, Mahdi F (2012) Identification of the c-kit gene mutations in biopsy tissues of mammary gland carcinoma tumor. J Egypt Natl Cancer Inst 24(2):97–103

    Article  Google Scholar 

  29. Ghasemzadeh-Hasankolaei M, Eslaminejad MB, Batavani R, Ghasemzadeh-Hasankolaei M (2015) Male and female rat bone marrow-derived mesenchymal stem cells are different in terms of the expression of germ cell specific genes. Anat Sci Int 90(3):187–196

    Article  CAS  PubMed  Google Scholar 

  30. Qin Q, Liu J, Ma Y, Wang Y, Zhang F, Gao S et al (2017) Aberrant expressions of stem cell factor/c-KIT in rat testis with varicocele. J Formos Med Assoc 116(7):542–548

    Article  CAS  PubMed  Google Scholar 

  31. Huang P, Lin LM, Wu XY, Tang QL, Feng XY, Lin GY et al (2010) Differentiation of human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells into germ‐like cells in vitro. J Cell Biochem 109(4):747–754

    CAS  PubMed  Google Scholar 

  32. Lacham-Kaplan O (2004) In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction 128(2):147–152

    Article  PubMed  Google Scholar 

  33. Kumar K, Das K, Madhusoodan A, Kumar A, Singh P, Mondal T et al (2018) Rat bone marrow derived mesenchymal stem cells differentiate to germ cell like cells. bioRxiv:418962

  34. Beltran M, Puig I, Peña C, García JM, Álvarez AB, Peña R et al (2008) A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial–mesenchymal transition. Genes Dev 22(6):756–769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cacheux V, Dastot-Le Moal F, Kääriäinen H, Bondurand N, Rintala R, Boissier B et al (2001) Loss-of-function mutations in SIP1 Smad interacting protein 1 result in a syndromic Hirschsprung disease. Hum Mol Genet 10(14):1503–1510

    Article  CAS  PubMed  Google Scholar 

  36. Yu M, Tian T, Zhang J, Hu T (2022) miR-141-3p protects against blood–brain barrier disruption and brain injury after intracerebral hemorrhage by targeting ZEB2. J Clin Neurosci 99:253–260

    Article  CAS  PubMed  Google Scholar 

  37. Lou G, Dong X, Xia C, Ye B, Yan Q, Wu S et al (2016) Direct targeting sperm-associated antigen 9 by miR-141 influences hepatocellular carcinoma cell growth and metastasis via JNK pathway. J Exp Clin Cancer Res 35(1):1–10

    Article  Google Scholar 

  38. Yadav RP, Kotaja N (2014) Small RNAs in spermatogenesis. Mol Cell Endocrinol 382(1):498–508

    Article  CAS  PubMed  Google Scholar 

  39. Kotaja N (2014) MicroRNAs and spermatogenesis. Fertil Steril 101(6):1552–1562

    Article  CAS  PubMed  Google Scholar 

  40. Abu-Halima M, Hammadeh M, Schmitt J, Leidinger P, Keller A, Meese E et al (2013) Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments. Fertil Steril 99(5):1249–1255e16

    Article  CAS  PubMed  Google Scholar 

  41. Su Y, Zhou LL, Zhang YQ, Ni LY (2019) Long noncoding RNA HOTTIP is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation. Mol Genet Genom Med 7(9):e870

    Google Scholar 

  42. Hadziselimovic F, Gegenschatz-Schmid K, Verkauskas G, Demougin P, Bilius V, Dasevicius D et al (2017) GnRHa treatment of cryptorchid boys affects genes involved in hormonal control of the HPG axis and fertility. Sex Dev 11(3):126–136

    Article  CAS  PubMed  Google Scholar 

  43. Flanagan CA, Manilall A (2017) Gonadotropin-releasing hormone (GnRH) receptor structure and GnRH binding. Front Endocrinol 8:274

    Article  Google Scholar 

  44. Lin J, Mao J, Wang X, Ma W, Hao M, Wu X (2019) Optimal treatment for spermatogenesis in male patients with hypogonadotropic hypogonadism. Medicine 98:31

    Google Scholar 

  45. Nishida H, Miyagawa S, Vieux-Rochas M, Morini M, Ogino Y, Suzuki K et al (2008) Positive regulation of steroidogenic acute regulatory protein gene expression through the interaction between Dlx and GATA-4 for testicular steroidogenesis. Endocrinology 149(5):2090–2097

    Article  CAS  PubMed  Google Scholar 

  46. Hadziselimovic F, Verkauskas G, Vincel B, Stadler MB (2019) Testicular expression of long non-coding RNAs is affected by curative GnRHa treatment of cryptorchidism. Basic Clin Androl 29:18

    Article  PubMed  PubMed Central  Google Scholar 

  47. Vigneau S, Rohrlich P-S, Brahic M, Bureau J-F (2003) Tmevpg1, a candidate gene for the control of Theiler’s virus persistence, could be implicated in the regulation of gamma interferon. J Virol 77(10):5632–5638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Collier SP, Collins PL, Williams CL, Boothby MR, Aune TM (2012) Cutting edge: influence of Tmevpg1, a long intergenic noncoding RNA, on the expression of Ifng by Th1 cells. J Immunol 189(5):2084–2088

    Article  CAS  PubMed  Google Scholar 

  49. Salviano-Silva A, Lobo-Alves SC, Almeida RC, Malheiros D, Petzl-Erler ML (2018) Besides pathology: long non-coding RNA in cell and tissue homeostasis. Noncoding RNA 4(1)

  50. Klein B, Haggeney T, Fietz D, Indumathy S, Loveland KL, Hedger M et al (2016) Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia. Hum Reprod 31(10):2192–2202

    Article  CAS  PubMed  Google Scholar 

  51. Gu J, Wang Y, Wang X, Zhou D, Shao C, Zhou M et al (2018) Downregulation of lncRNA GAS5 confers tamoxifen resistance by activating miR-222 in breast cancer. Cancer Lett 434:1–10

    Article  CAS  PubMed  Google Scholar 

  52. Li M, Xie Z, Wang P, Li J, Liu W, Liu Z et al (2018) The long noncoding RNA GAS5 negatively regulates the adipogenic differentiation of MSCs by modulating the miR-18a/CTGF axis as a ceRNA. Cell Death Dis 9(5):1–13

    Article  Google Scholar 

  53. Yacqub-Usman K, Pickard MR, Williams GT (2015) Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells. Prostate 75(7):693–705

    Article  CAS  PubMed  Google Scholar 

  54. Wang J, Gong X, Tian GG, Hou C, Zhu X, Pei X et al (2018) Long noncoding RNA growth arrest-specific 5 promotes proliferation and survival of female germline stem cells in vitro. Gene 653:14–21

    Article  CAS  PubMed  Google Scholar 

  55. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY et al (1999) A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97(1):17–27

    Article  CAS  PubMed  Google Scholar 

  56. Yan R, Wang K, Peng R, Wang S, Cao J, Wang P et al (2016) Genetic variants in lncRNA SRA and risk of breast cancer. Oncotarget 7(16):22486

    Article  PubMed  PubMed Central  Google Scholar 

  57. Hube F, Velasco G, Rollin Jm, Furling D, Francastel C (2011) Steroid receptor RNA activator protein binds to and counteracts SRA RNA-mediated activation of MyoD and muscle differentiation. Nucleic Acids Res 39(2):513–525

    Article  CAS  PubMed  Google Scholar 

  58. Xu B, Yang W-H, Gerin I, Hu C-D, Hammer GD, Koenig RJ (2009) Dax-1 and steroid receptor RNA activator (SRA) function as transcriptional coactivators for steroidogenic factor 1 in steroidogenesis. Mol Cell Biol 29(7):1719–1734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Luo P, Jing W, Zhu M, Li N-D, Zhou H, Yu M-X et al (2017) Decreased expression of LncRNA SRA1 in hepatocellular carcinoma and its clinical significance. Cancer Biomark 18(3):285–290

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Thanks to Dr. Mostafa Latifpour and his colleagues to provide the Human Wharton Jelly Mesenchymal Stem Cells as a gift.

Funding

This study was derived from a thesis for an M.Sc. degree in the field of medical biotechnology (Grant or Code Number: 950214021) at Gorgan School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran.

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Authors and Affiliations

Authors

Contributions

SGH: Performing experiments, acquisition of data, analysis and interpretations of data, manuscript drafting, revision of the manuscript. MSH: Contribution to study design. GAF: Contribution to data analysis and manuscript drafting. JTA: Contribution to the evaluation of experiments and manuscript drafting. MS: Contribution to study design. SR: Contribution to manuscript revision. NM: Study design and concept, participation in literature bibliography, data acquisition and analysis, manuscript drafting, and critical revision of the manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Naser Mobarra.

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The study was approved by the Committee of Ethics, Golestan University of Medical Sciences (Code of Ethics: IR.GOUMS.REC.1394.34).

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The authors declare that they have no conflicts of interest.

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Ghasemi, S., Shafiee, M., Ferns, G.A. et al. Differentiation of Human Wharton Jelly Mesenchymal Stem Cells into Germ-Like Cells; emphasis on evaluation of Germ-long non-coding RNAs. Mol Biol Rep 49, 11901–11912 (2022). https://doi.org/10.1007/s11033-022-07961-6

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